This application relates to a method of generating, screening and identifying small disulfide rich conopeptides having high affinity and specificity for selected target proteins using cloning, and particularly, fusion phage technology.
The marine gastropod mollusks belonging to the superfamily Conacea, (also called Toxoglossa) are distinguished by the general presence of a toxin gland and a hollow tooth for delivery. This superfamily, comprising approximately 4000 species, perhaps 10% of all molluscan species diversity, is one of the most successful of all marine taxa. There are three main groups or families, i.e. the cone (Conus), the tower shells (Terebra) and the slit shells (Turris). All species are predators, the vast majority using venom as the primary means for subduing prey.
One factor that contributes to the success of this group is the remarkable biochemistry of their venoms. Conacea venom may be particularly important to understand in all of its facets. The biologically active components of at least some of these venoms are an unprecedented diversity of small, conformationally rigid peptides having specifically located Cys residues and multiple disulfide bonds. These peptides are amenable to known methods for chemical synthesis and structure determination. In addition, they are directly translated from genes, and can therefore be manipulated by state of the art molecular biological technologies.
Most information developed thus far, including biochemical analysis of venoms, have been done on species of the Conus family. However, a direct biochemical analysis of the venoms from the various species of the Terebra and Turnis families has not yet been carried out. The roughly 500 species of the Conus family can be divided into three groups on the basis of the prey they feed on, i.e. the worm-hunting, mollusc-hunting and fish-hunting species. Venoms of at least one species of each of these groups has been analyzed and the major biologically active components have been identified as small peptides, typically of about 10 to 30 amino acids in length. A particular Conus venom may have well over 50 different small peptides, many of them with different pharmacological specificity. From the numerous peptides which have been purified from various Conus species, a number of generalizations have emerged which form the basis for the presently claimed invention. The peptides isolated from venom of all the various species within the Conacea superfamily are generically referred to in this description as "conotoxins".
Each conotoxin peptide appears to be specifically targeted to a macromolecular receptor, interfering with its normal function. The conotoxins can have very high affinities for their receptors. In the case of certain .omega.-conotoxins, for example, subpicomolar affinities are achieved for their high-affinity Ca++ channel targets. A key feature that contributes to the high affinity and specificity of the conotoxin peptides is attributed to their fixed, relatively rigid conformation. Normally, peptides in the 10 to 30 amino acid range would not have a specific fixed conformation because of their small size. Under most physiological conditions, it takes a polypeptide of about 40-50 amino acid residues before the sum total of non-covalent forces, (hydrogen bonds, hydrophobic interactions, and the like) is sufficient for a specific conformation to be stable. The vast majority of the conotoxin peptides are conformationally restrained by covalent cross-linking through multiple disulfide bonding. Typically between about 20 to 50% of all amino acids in a conotoxin peptide are Cys residues. Conotoxin peptides have some of the highest known densities of disulfide bonding in any biological system. Peptides from Conus venoms have been isolated that are only 12 amino acids long with three disulfide bonds. In general, only one disulfide bonded configuration of a particular toxin exhibits high affinity for the specific receptor target. Although some conotoxins have only two disulfide bonds (notably the .alpha.-conotoxins which target to nicotinic acetylcholine receptors), more commonly the major paralytic conotoxins found in Conus venom have three disulfide bonds (in the fish-hunting cones, the .mu.-conotoxins which target to muscle voltage-sensitive Na channels and the .omega.-conotoxins which target to presynaptic voltage sensitive Ca channels). For the latter, there are 16 possible disulfide bonded configurations. Folding smaller peptides into one specific configuration is a biochemical problem which the cone snails had to solve before they could efficiently use such small peptides as high affinity ligands for paralysing their prey.
Small, conformationally constrained peptides are ideal for a wide variety of biotechnology applications. Their small size facilitates access to specific target receptors. Specific cross-linking of disulfide bonds allows these small peptides to assume a relatively rigid structure that increases the probability of high affinity interaction with target molecules. Still, the variation of peptide structure afforded by variation in amino acid sequence in such peptides is enormous. Natural variants among peptides following this architectural design have been found to target a great diversity of target types.
One feature that has emerged from sequencing many conotoxin peptides is that most of them fall into characteristic patterns of disulfide forming Cys residue arrangements. The major, or standard framework, patterns are represented by the following formulas with Cys being represented by "C" and "--" representing a variable grouping of other amino acids:
______________________________________ "2-loop" framework CC--C--C (Formula I) Seq. ID NO: 15! "3-loop" framework CC--C--C--CC (Formula II) Seq. ID NO: 16! "4-loop" framework C--C--CC--C--C (Formula III) Seq. ID NO: 17! ______________________________________
These characteristic Cys arrangements are found as a dominant structural feature in conotoxin peptides from all three feeding classes of the Conus family and may be present in the venoms of all families within the Conacea superfamily. The conotoxin peptide sequence information from mollusk-hunting venoms is typical. Of 17 peptides sequenced sufficiently for assessing the Cys residue arrangement, two peptides had the standard 2-loop configuration (Formula I), five had the standard 3-loop structure (Formula II) and nine had the standard 4-loop framework (Formula III). One peptide did not fit a standard framework and had a minor 5-loop configuration. The actual disulfide bonding in at least one member of each of the standard frameworks has been determined. The disulfide bonds were formed between the first and third and second and fourth Cys residues in the 2-loop structure and between the first and fourth, second and fifth and third and sixth residues respectively in the 3-loop and 4-loop frameworks.
It is hypothesized that the standard loop structures are established in the conotoxins because the Conus snails have evolved genetic mechanisms for efficiently and rapidly generating variant sequences, mechanisms specifically geared to using the standard Cys frameworks. In this way, the Conus snails are able to achieve a remarkable sequence plasticity in their peptides and a corresponding pharmacological diversity even though the same Cys frameworks, and hence, disulfide bond configurations, are used again and again. For example, the major group of conotoxins which target to Na channels have a standard 3-loop framework but another conotoxin targeting to the same Na channel has the standard 4-loop framework. The two peptides have no apparent homology and appear to have evolved independently. In addition, 4-loop Ca channel targeted peptides, with no affinity for Na channels and no homology to the 4-loop Na channel peptide are present in the same venom. A single venom from a Conus snail comprises numerous small peptides, most of which exhibit standard 2-, 3- and 4-loop Cys frameworks. Despite the small numbers of different Cys frameworks, the diversity of peptides in any given Conus venom is truly remarkable. Moreover, each of the thousands of species of the Conacea superfamily may have its own distinctive complement of peptide sequences. In the data obtained from ten species of the Conus family, no single peptide sequence has been found to occur in more than one venom. Therefore, it is believed that there are many thousands of peptides in the Conacea venoms each having its own distinctive pharmacological properties. As illustrative, one may consider the .alpha.-conotoxins which are the major post-synaptic paralytic toxins targeting to acetylcholine receptors. It has been found that each fish-hunting species has its own set of .alpha.-conotoxins, having sequences different from any other species.
The pharmacological complexity of a single venom may be illustrated by examining the in vivo effects of injecting each peptide from one size fraction of a single Conus geographus venom. This experiment is described in detail in Olivera et al., (1990) Science 249, pp. 217-232. The venom was eluted into fractions on a Sephadex G-25 column. One major fraction was further separated by HPLC using a VYDAC C18 column and a trifluoroacetic acid-acetonitrile gradient. The elution profile showed numerous peaks when absorbance at 210 nm is plotted as a function of elution time. Each peak, consisting of either a single peptide or mixture of several peptides, was tested for biological activity by injecting from 0.5 to 2 nmol intracranially into mice and the CNS response elicited was recorded. In many cases the activity observed is either the symptomatology induced by the most potent component or a composite of symptoms from several peptides. Whether the activity is the result of a single conotoxin or mixture of conotoxins the following responses were observed, each coming from a single peak in this one size fraction, (1) head swinging, (2) circular motion, (3) dragging back legs, (4) sleeper/climbing, (5) uncoordinated, (6) twisted jumping, (7) paralysis, (8) kicking on back and scratching, (9) depressed activity, (10) comatose (lethal to at least one animal), (11) paralysis (lethal to at least one animal), (12) depressed activity, (13) trembling, (14) dragging (lethal to at least one animal), (15) depressed followed by hyperactivity (lethal to at least one animal), (16) normal, (17) scratching and convulsion (lethal to at least one animal), (18) convulsion and bleeding (lethal to at least one animal), (19) convulsion (lethal to at least one animal) and (20) normal.
It is apparent from the above that the conotoxins have distinctive properties which make them of particular interest for biotechnological applications. Although there is a remarkable pharmacologic diversity of conotoxins, each peptide is specifically targeted. In certain cases, the conotoxins are able to discriminate between closely related subtypes of a receptor target.
One reason for some of the unique properties of the conotoxins may well be a response to the relentless selection for rapid paralysis. The unusually small size of the conotoxin peptides may have evolved to facilitate the efficient dissemination of the toxins through the body of the prey. A small peptide of 10-30 amino acids will cross permeability barriers (such as the blood vessels of fish) much more quickly than a typical 50-90 amino acid proteinaceous toxin of snakes or scorpions. In this respect, the conotoxins can literally immobilize the prey 1-2 seconds after injection of the venom and effect complete paralysis a few seconds later.
Molecules of this type have an expanding usefulness as agents capable of targeting a vast variety of receptors and ion channels on the surface of many different cell types. These molecules will be useful in the design and testing of drugs targeting a variety of therapeutically important receptors, and in the design of agriculturally important agents such as pesticides. These peptides are unique ligands which potentially affect the function of their target proteins.
The major object of this invention is the de novo, snail-free generation of conotoxin-like peptides. Although the natural complement of conotoxins has many potential applications, the ability to produce novel peptides in vitro with predetermined target specificity would vastly expand this potential. It has been shown by Olivera et al., (1990) Science, 249, pp 217-232, which is incorporated herein by reference, that this general class of rigid, mulitple disulfide-linked peptides can be ligands of exquisitely refined specificity. At this time, no conotoxin-like peptide has yet been generated in vitro with a novel specificity. The invention described is drawn to a means of producing ligands with the same general characteristics as the natural conotoxins, i.e., peptides with high affinity and specificity for a target receptor, which can be chemically synthesized, and that have a relatively rigid conformation due to multiple disulfide bonds. When such peptides are bound to their receptor target, they affect the biological activity of that target. Such conotoxin-like peptides will, in this description, be called either "conoeffector peptides" or "conopeptides", terms which will be used interchangeably. Conoeffector peptides with particular biological activity will be identified by screening a general conopeptide library for clones which bind to a particular receptor protein and affect its biological activity. The conoeffector peptides so identified will yield sufficient chemical-structure information to allow peptidomimetic drug design. In addition, further rounds of screening could yield conopeptides with still more refined receptor or phylogenetic specificities.
Although the natural spectrum of peptides provided by the conotoxins is highly diverse, specific application will require peptides with receptor or phylogenetic specificities which may not be found directly in the set of natural conotoxin peptides. For example, in the field of pesticides, it may be desirable to have toxins that specifically kill only one order or insects, but do not affect other arthropods, nor animals in other phyla. Such specificity is unlikely to be found in the set of natural conotoxins because they are targeted in vivo either to vertebrates, mollusks, or three phyla of worms, and not to insect receptors. However, in principle, it should become possible to carry out phylogenetic focusing in vitro. Once a peptide structure has been found which targets to a particular target protein or receptor type, it should be possible to use molecular genetics to select variants with the desired phylogenetic specificity. Thus, in the case of insecticides, the lead structure might be a "broadly focused" or even a phylogenotically non-discriminating conotoxin structure which acts on the target insect. A library could then be constructed, each containing a variant of the lead conotoxin sequence. Variants can be selected to bind the target insect receptor, but show no binding to homologous receptors in other taxa.
The same reasoning will also apply to the design of conopeptides having other biological and/or pharmacological applications.
Several potential applications of in vitro-generated conoeffector peptides are based on the potential ability of the conopeptides to distinguish between closely related subtypes of a particular receptor target. A major problem in drug design, for example, is to rationally minimize side effects. In recent years, recombinant DNA technology has revealed that multiple subtypes exist for practically every cell-surface receptor involved in intercullular signaling and signal transduction. Although a drug may be targeted to a particular therapeutically relevant receptor target, it may interact with closely related subtypes as well. The latter interactions are the probable cause of the side effects of many drugs. Conceptually, the most straightforward way to circumvent this problem is to develop agents that only interact with therapeutically relevant receptor subtype targets with no cross reactivity to related receptor subtypes.
Conotoxin structures clearly have the potential for such refined pharmacological targeting. It has been demonstrated that certain conotoxins can exhibit remarkable receptor subtype discrimination. In particular, the .omega.-conotoxins, which target to calcium channels, show a tissue specificity with regard to calcium channels that is exhibited by no other ligand that has been designed by the pharmaceutical industry so far. Thus, the dihydropyridine drugs will affect calcium channels not only in skeletal muscle, cardiac muscle and smooth muscle but in neuronal tissue as well. In contrast, the .omega.-conotoxins target to certain neuronal calcium channel subtypes and show a &gt;10.sup.8 fold discrimination index against calcium channels which are non-neuronal in the most favorable cases. This remarkable ability to discriminate between what are probably closely related receptors demonstrates that the basic conotoxin structural motifs are capable not only of having very high affinity for a particular receptor subtype, but a high discrimination index against closely related subtypes.
In principle, conoeffector peptides can be generated in vitro which affect the activity of the desired subtype of a particular receptor, but are screened not to bind other closely related subtypes at all. Therefore, this invention has the potential to provide the pharmaceutical industry with a rational methodology for designing drugs with minimal undesirable side effects. In principle, it should be possible to design an .omega.-conotoxin-like molecule that only is targeted to a single calcium channel subtype with no cross reaction against any other calcium channel, nor other receptors. This has great potential application, since calcium channels control many specific properties of nerve cells, including neurotransmitter release. Other specific applications include drugs for Parkinson's disease, schizophrenia and depression. The side effects of many therapeutically effective drugs for these conditions is a continuing problem. Some of the most effective drugs affect the metabolism of the neurotransmitters dopamine and serotonin. The biological effects of these neurotransmitters have been shown to be mediated through dopamine receptors and serotonin receptors, both of which are present as multiple subtypes, encoded by multiple genes. Many of these receptor subtypes have recently been cloned, and can thus be used to screen conopeptide libraries. The possibility of obtaining a small rigid peptide specifically targeted for agonist or antogonist activity to each dopamine receptor subtype would have obvious implications in drug development for Parkinsonism and schizophrenia.
The basis for using an in vitro approach for the focusing of conopeptides to specific protein targets requires a system into which conopeptide modules can be cloned. The sequence of the conopeptide module can be varied by inserting oligonucleotides with random nucleotide sequences in selected positions. The variant conopeptide sequences could then be screened for binding to receptors or other selected targets. Preferably the conopeptides will be cloned such that they are displayed on the surface of a carrier protein or in a cell compartment where they can be exposed and, if active, bound to a specified receptor or other target protein molecules.
A system wherein other peptides have been successfully cloned and screened for binding specificity has recently been described by two of the inventors herein and is referred to as "fusion phage", e.g. see Scott et al., Searching for Peptide Ligands with an Epitope Library, Science, 249, 386-390 (Jul. 20, 1990) which is incorporated herein by reference. "Fusion phage" are filamentous bacteriophage vectors in which foreign sequences are cloned into phage gene III and displayed as part of the gene III protein (pIII) at one tip of the virion.
In the fusion phage techniques of Smith et al., supra, a library was constructed of phage containing a variable cassette of six amino acids. The variable hexapeptide modules, when fused to the bacteriophage, provides a library for the screening methodology that can examine &gt;10.sup.12 phages (or about 10.sup.8 -10.sup.10 different clones) at one time, each with a test sequence on the virion surface. The library obtained was used to screen monoclonal antibodies specific for particular hexapeptide sequences. The fusion phage system has also been used by other groups and libraries containing longer peptide inserts have been constructed as shown by Devlin et. al., (1990) Science 249, 404-406 and libraries displaying antibody variable domains are shown by McCafferty et.al., (1990) Nature 348, 552-554. The peptides, such as referred to by Smith et al. and Devlin et al., supra, have a disadvantage in the bacteriophage screening technique since they do not have rigid structure and usually have longer, flexible amino acid chains. In such long chain flexible peptides, potential interactive sites may tend to be masked by the folding of the peptide chain, thereby making it almost impossible to screen the library effectively. In addition, because the peptide inserts suggested by these articles are relatively flexible, the binding conformation may be assumed as only one of many alternative and thus the peptide module may not bind to a receptor site with as great affinity as is desirable. Indeed, the hexapeptide library of Scott et al., supra, has not produced binding clones for five different receptors used Scott, (1991) Trends in Biochemistry, (in press)!.
However, the conopeptide type molecules should be well suited for this type of screening since they have multiple disulfide bonds which give them a rigid structure that binds readily with the reactive sites of the receptors or other target protein molecules. Hundreds of millions of different conopeptides could be easily surveyed for tight binding to a receptor or other binding protein using a cloned library of conopeptides.
It would therefor be desirable to combine the structural information gleaned from the conotoxins with fusion phage technology in the design of such "conoeffector peptide" libraries. Such libraries would provide necessary information for identifying clones of phage bearing conopeptides which bind specifically, and with high affinity to a particular receptor and other target proteins, and affect their function. Once a clone has been isolated and identified as binding tightly to receptor, the encoded conoeffector peptide can be synthesized. Such receptor-targeted conopeptides could be used to obtain sufficient chemical-structural information to allow the design of any variety of pharmaceuticals, pesticides, or other bioactive agents limited only by the availability of appropriate receptor or other target protein molecules.